Serotonin neurons of the raphe nuclei control mood and are sensitive to drugs of abuse such as the amphetamine MDMA (3,4-Methylenedioxymethamphetamine, Ecstasy) and cocaine. Because serotonin release by axon-derived varicosities, cell bodies and dendrites cannot be assayed and compared directly in intact tissue, the contribution of each cellular compartment to release evoked by electrical activity and drugs has not been quantified. Likewise, the properties of release sites near a dorsal raphe nucleus serotonin neuron that arise from that neuron, from neighboring neurons in the same nucleus and projections from other raphe nuclei have not been determined with current methods. Therefore, we have been developing a new optical technique for studying serotonin neurons in brain slices. Multiphoton monoamine imaging (MMI) utilizes a fluorescent serotonin analog that is excited by 725 nm pulsed infrared light from Ti:sapphire laser. We have demonstrated that MMI detects uptake and depolarization-evoked release from serotonin neuron cell bodies and varicosities in dorsal raphe nucleus brain slices. Here, experiments are presented to establish the utility of MMI for studying release in brain slices induced by relevant stimuli (action potentials and MDMA) at identified compartments (axonal boutons, dendrites and cell bodies) of serotonin neurons. This further development will ensure that MMI will be able to address fundamental questions concerning the effects of abused drugs on serotonin neurons. PUBLIC HEALTH RELEVANCE: Drug abuse continues to be a major public health problem. It is known that the neurotransmitter serotonin affects mood (e.g. depression, anxiety) and dopaminergic reward systems involved in addiction. This CEBRA application will further develop a new optical method called Multiphoton Monoamine Imaging for studying transmitter release by serotonin neurons.